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Methods, articles, and compositions for identifying oligonucleotidesMethods, articles, and compositions for identifying oligonucleotides description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20080050718, Methods, articles, and compositions for identifying oligonucleotides. Brief Patent Description - Full Patent Description - Patent Application Claims
II. BACKGROUND [0002]2. There are many situations where oligonucleotides that efficiently bind a target DNA or RNA are desired. These oligonucleotides can be used for a variety of purposes, including antisense, diagnostics, and array generation. While researchers have worked for many years to identify algorithms and methods for predicting the oligonucleotides that will bind the target with the highest efficiency, better prediction methods are needed. Disclosed are methods, articles, machines, and compositions that aid in identifying oligonucleotides and sets of oligonucleotides that will efficiently bind a target nucleic acid molecule. Also disclosed are optimized sets of oligonucleotides that bind HIV-l genomic RNA or DNA,, such as the GAG RNA, and methods of using them. III. SUMMARY [0003]3. Disclosed are methods and compositions related to methods, compositions, and articles related to identification of oligonucleotides designed to hybridize with a target nucleic acid. IV. BRIEF DESCRIPTION OF THE DRAWINGS [0004]4. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods. [0005]5. FIG. 1 shows a scheme of oligonucleotide-target RNA interaction, which shows thermodynamic factors that can influence oligonucleotide RNA hybridization intensity. [0006]6. FIG. 2 shows an RNA hybridization intensity profile for the set of oligonucleotides (20 mers) that was used for creation of the first dataset. The hybridization intensity is shown for each oligonucleotide in relation to its position in the target RNA. For statistical analysis, the oligonucleotides were categorized into groups according to hybridization intensity. The small arrow represents the group with low hybridization intensity; medium sized arrow, intermediate; and large arrow with high. [0007]7. FIG. 3 shows a relationship between calculated thermodynamic parameters and hybridization intensity of the oligonucleotides with their target RNA. [0008]8. FIG. 4 shows a categorization of oligonucleotides into subsets according to their thermodynamic properties. The percentage of oligonucleotides with RNA hybridization intensity higher than the defined threshold in each subset is shown. The code is the same as in FIG. 2. Numbers of oligonucleotides in each subgroup are printed on highlighted parts of the columns. The proportion of oligonucleotides in each subset versus the total number of oligonucleotides in the relevant dataset is shown above each column. Subset 1 contains oligo-probes that can form stable duplexes with RNA dG.degree..sub.25.ltoreq.29 kcal/mol; subset 2 contains the oligo-probes that can form stable duplexes with RNA dG.degree..sub.25.ltoreq.29 kcal/mol with unstable intermolecular oligo self-structures dG.degree..sub.25.gtoreq.8 kcal/mol; and subset 3 contains oligo-probes that can form stable duplexes with RNA dG.degree..sub.25.ltoreq.29 kcal/mol but which form both unstable inter- and intra-molecular self-structures (dG.degree..sub.25.gtoreq.8 kcal/mol for inter-molecular structures and dG.degree..sub.25.gtoreq.1.1 kcal/mol for intra-molecular structures). [0009]9. FIG. 5 shows a relationship between thermodynamic evaluations of oligonucleotide inter- and intra-molecular pairing potentials (x andy axes, respectively). Medoum gray squares represent the group with low hybridization intensity; light gray, intermediate; and dark grey with high. [0010]10. FIG. 6 shows a categorization of oligonucleotides into subsets according to their thermodynamic properties. Two sets of oligonucleotides in dataset 2 are shown. The first set represents all oligonucleotides in the dataset, while the second represents only the fraction with certain thermodynamic properties. The proportion of oligonucleotides in each subset versus the total number of oligonucleotides in dataset 2 is shown above each column. The percentage of oligonucleotides with RNA hybridization intensity higher than the defined threshold in each set is also shown. The code is the same as in FIG. 2. Numbers of oligonucleotides in each subgroup are printed on highlighted parts of the columns. Subset 4 contains oligo-probes that can form stable duplexes with RNA dG.degree..sub.25.ltoreq.35 kcal/mol but which form both unstable inter- and intra-molecular self-structures (dG.degree..sub.25.gtoreq.8 kcal/mol for inter-molecular structures and dG.degree..sub.25.gtoreq.1.1 kcal/mol for intra-molecular structures). [0011]11. FIG. 7 shows a relationship between calculated values of dG.degree..sub.25 of DNA-RNA duplex stability and hybridization intensities of the oligonucleotides with their target RNA for the subset of oligo-probes with little self-structure from dataset 3. [0012]12. FIG. 8 shows a scheme for evaluation of cross-hybridization potentials of oligo-probe candidates. [0013]13. FIG. 9 shows scatter plots showing the relationship between thermodynamic parameters and antisense oligonucleotide activities from both databases. Activity values (A) are expressed as the ratio of the level of a particular mRNA or protein measured in cells treated with an antisense oligonucleotide, to the level of the same mRNA or protein in untreated cells. Linear or non-linear trend lines are shown in each scatter plot. [0014]14. FIG. 10 shows a relationship between thermodynamic parameters and antisense oligonucleotide activities determined for the web database. (A) Oligo nucleotides were categorized into two groups according to calculated values of dG.degree..sub.37 for DNA-RNA duplex formation. Group 1 contains oligonucleotides that form more stable duplexes, and group 2 contains oligonucleotides that form less stable duplexes with target RNA. (B) Group 1 oligonucleotides separated on the basis of the calculated dG.degree..sub.37 for oligonucleotide intra-molecular pairing. (C) Group 1 oligonucleotides separated on the basis of the calculated dG.degree..sub.37 for oligonucleotide inter-molecular pairing. The numbers of oligonucleotides in each subgroup are indicated in the relevant highlighted segments. [0015]15. FIG. 11 shows a relationship between thermodynamic parameters and antisense oligonucleotide activities determined for the Isis database. Oligonucleotides were categorized into two groups according to the calculated value of dG.degree..sub.37 of duplex formation. (A) Group 1 contains oligonucleotides that form more stable duplexes and group 2 contains oligonucleotides that form less stable duplexes with target RNA. (B) Group 1 oligonucleotides were further separated based on the calculated dG.degree..sub.37 for oligonucleotide intra-molecular pairing. (C) Group 1 oligonucleotides were further separated based on the calculated dG.degree..sub.37 for oligonucleotide inter-molecular pairing. For each set, oligonucleotides were separated into subgroups according to their antisense efficacy. The numbers of oligonucleotides in each subgroup are on the relevant highlighted segments. [0016]16. FIG. 12 shows a relationship between thermodynamic evaluations of oligonucleotide inter- and intra-molecular pairing potentials (x- and y-axis, respectively). The trend line is shown in each scatter plot. [0017]17. FIG. 13 shows a relationship between thermodynamic parameters and antisense oligonucleotide activities from both databases. (A) Data from the published antisense oligonucleotide experiments. (B) Unpublished data from Isis Pharmaceuticals. The numbers of oligonucleotides in each subgroup are on the relevant segments. Set 1 contains all oligonucleotides in each database. Set 2 includes only oligonucleotides predicted to form very stable duplexes (dG.degree..sub.37.ltoreq.30 kcal/mol) and those with the least possibility for self-structure (dG.degree..sub.37.gtoreq.5 kcal/mol for inter-molecular oligonucleotide pairing and dG.degree..sub.37.gtoreq.1 kcal/mol for intra-molecular pairing). [0018]18. FIG. 14 shows a consensus GAG sequence and a plot of conservation with a 30 nucleotide window. FIG. 14A shows Gag consensus sequence. Last nucleotides in the theoretically optimal target regions are highlighted. The range of fragments that were analyzed was from 23 to 35-mers. The length of optimal region is shown below the highlighted nucleotide. Only numbers for shortest regions in the sets that correspond to each highlighted nucleotide are shown. FIG. 14B shows a Gag plot of conservation made with window of 30 nucleotides and step 1. Average conservation for each consequent 30 nucleotides is shown. Conserved regions that are thermodynamically optimal for oligonucleotide targeting are highlighted. [0019]19. FIG. 15 shows the number of theoretically optimal RNA targets obtained with each possible length of oligonucleotide, in the range from 23 to 35-mers. V. DETAILED DESCRIPTION [0020]20. Disclosed are methods, compositions, and articles that allow for the efficient identification of oligonucleotides that will hybridize better with target sequences. These methods, compositions, and articles are based on the disclosed understanding of certain thermodynamic parameters and how they relate to each other and how they affect the efficient binding of a given oligo for a target nucleic acid. One nucleic acid binds or hybridizes with another nucleic acid based on the ability of the two nucleic acids to form base pairs with each producing a duplex or double stranded DNA molecule. Whether two nucleic acids hybridize is a combination of the thermodynamic properties of four separate interactions that take place or can take place between the first nucleic acid or oligo, for example, and the second nucleic acid, or target. These four parameters are shown in FIG. 1. The first parameter is the Gibbs free energy, delta G, or dG of the interaction between the oligo and the target RNA molecule. This is the dG of the desired interaction, or the sub part of the total energy that arises when the oligo and the target come together that is due to the actual interactions between the oligo and the target. This parameter can be represented as dG.degree..sub.oligo-RNA duplex. Another parameter that can effect the overall dG of the target and oligo coming together is the self structure of the oligo itself, the ability of the oligo to form secondary and tertiary structures, such as hairpins or pseudoknots. This parameter can be represented as dG.degree..sub.oligo-structure. A third parameter that can effect the overall dG for the oligo-target interaction is the dG of the oligo forming dimers or multimers with itself. This third parameter can be represented as dG.degree..sub.oligo-oligo dimer. Lastly, the fourth parameter that can effect the overall dG of oligo and target is the self structure of the target RNA molecule itself. This fourth parameter can be represented as dG.degree..sub.RNA structure. It is understood that the dG.degree..sub.oligo-RNA duplex can be considered a promotion force behind the overall force bring the oligo and the target together and that the dG.degree..sub.oligo-structure, dG.degree..sub.oligo-oligo dimer, and dG.degree..sub.RNA structure can be considered negative forces, in essence reducing the ability of the oligo and target to come together. These parameters are in essence competing energies for the energy of duplex formation. Oligo intra- or inter-molecular structure can compete with oligo-target duplex formation and result in low hybridization intensity. Extensive secondary structure of the target can also limit this efficiency. Disclosed herein it is shown that thermodynamic considerations of the relative stability of oligo-target duplexes and both oligo intra- and inter-molecular self-structures, without consideration of target secondary structure, can be sufficient for selection of oligo-probes that are efficient target binders. In other embodiments the structure of the target nucleic acid can also be considered. The disclosed methods, articles, and compositions, are provide guidelines for how to weight each of these parameters and how to analyze a given oligo's likelihood of being an oligo having a relatively strong overall affinity for a target nucleic acid molecule, such as an RNA molecule. Disclosed are methods that allow for the identification of sets of oligos that will have a higher probability of having a better overall affinity for binding the target nucleic acid. Also disclosed are compositions and articles, as well as machines that can be used in the disclosed methods. In certain embodiments, general methods that allow for the identification of any oligo for a specific target region are disclosed. In addition, methods that allow for the identification optimal oligos for a target even when the target has varying regions are disclosed. [0021]22. In certain embodiments the disclosed methods are designed for identifying oligos that bind at set temperatures, such as 37.degree. C. or 25.degree. C. Furthermore, in certain methods, the design is for conditions where there is higher ionic strength, for example, higher than the ionic strength of a typical PCR reaction and at relatively low temperatures, for example, under about 65.degree. C. This is because existing methods that predict effective oligonucleotide primers for identifying primers for these other conditions, such as picking primers for PCR reactions for a particular DNA template, work well for those applications because the primers will be employed under relatively stringent conditions. Thus PCR experimental primer design greatly simplifies the prediction problem: hybridization is performed at relatively low ionic strength and high temperature. Under these relatively stringent conditions, oligonucleotide and target secondary structures and oligo-oilgo duplex/multimer formation (dG.degree..sub.oligo-structureddG.degree..sub.RNA structure, and dG.degree..sub.oligo-oligo dimer are relatively unimportant. However, as discussed herein these structures become much more important at temperatures closer to and around 37.degree. C. These lower temperatures of oligo-RNA hybridization are frequently used in a number of different RNA detection assays and so efficient prediction of preferred oligo sets are desired. The disclosed methods, compositions, and articles, are designed to increase the efficiency of oligonucleotide design for target hybridization at around 37.degree. C. Methods for identifying the optimal parameters for a given temperature are known and can be found in U.S. patent application Ser. No. 10/374,253, filed on Feb. 26, 2003, for "Methods for designing oligo-probes with high hybridization efficiency and high antisense activity" by Olga Matveeva, and which is herein incorporated by reference in its entirety and at least for material related to methods for determining the threshold levels for the thermodynamic parameters at any given temperature and for material related to the identification and use of these parameters. Continue reading about Methods, articles, and compositions for identifying oligonucleotides... Full patent description for Methods, articles, and compositions for identifying oligonucleotides Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Methods, articles, and compositions for identifying oligonucleotides patent application. 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